Persistent gaseous bubbles as ultrasound contrast media

ABSTRACT

Disclosed herein are agents for enhancing the contrast in an ultrasound image. These agents are extremely small bubbles, or &#34;microbubbles,&#34; comprised of specially selected gases. The microbubbles described herein exhibit long life spans in solution and may be produced at a size small enough to traverse the lungs, thus enabling improved ultrasound imaging of the cardiovascular system and other vital organs. Also disclosed herein is a method for selecting gases from which contrast agents may be produced. The method is based on calculations using inherent physical properties of gases and describes a means to associate the properties of a gas with the time for dissolution of a microbubble comprised of the gas.

This is a divisional of application Ser. No. 08/071,377, filed Jun. 4,1993, now U.S. Pat. No. 5,393,524, which is a file wrapper continuationapplication of U.S. Ser. No. 074/761,311, filed on Sep. 17, 1991, nowabandoned.

DESCRIPTION

This invention relates to agents that enhance the contrast in anultrasound image generated for use in medical diagnosis. Thecontrast-enhancing media disclosed herein are comprised of extremelysmall gas bubbles which are present in a solution that is infused intothe body during or just before an ultrasound image is generated. Thisinvention is also directed to a method for enhancing such images byselecting gases from which a collection of free gas microbubbles can beprepared that have novel and superior properties. These microbubbles,composed of the gases whose selection is enabled by the process of thisinvention, may be extremely small in size and yet survive in thebloodstream long enough to allow contrast-enhanced imaging of parts ofthe cardiovascular system, peripheral vascular system, and vital organspreviously believed to be inaccessible to free gas microbubbles.

BACKGROUND

When using ultrasound to obtain an image of the internal organs andstructures of a human or animal, ultrasound waves--waves of sound energyat a frequency above that discernable by the human ear--are reflected asthey pass through the body. Different types of body tissue reflect theultrasound waves differently and the reflections, often aptly describedas "echoes," that are produced by the ultrasound waves reflecting offdifferent internal structures are detected and converted electronicallyinto a visual display. This display may prove invaluable to a physicianor other diagnostician in several ways, including evaluating theprogression of cardiovascular disease or the existence or nature of atumor.

For some medical conditions, obtaining a useful image of the organ orstructure of interest is especially difficult because the details of thestructure may not be adequately discernible from the surrounding tissuein an ultrasound image produced by the reflection of ultrasound wavesabsent a contrast-enhancing agent. Additionally, traditional ultrasoundimages are notoriously poor in quality and resolution. For thesereasons, detection and observation of certain physiological conditionsmay be substantially improved by enhancing the contrast in an ultrasoundimage by infusing an agent into an organ or other structure of interest.In other cases, detection of the movement of the contrast-enhancingagent itself is particularly important. For example, a distinct bloodflow pattern that is known to result from particular cardiovascularabnormalities may only be discernible by infusing a contrasting agentinto the bloodstream and observing the dynamics of the blood flow.

Medical researchers have made extensive investigation into the use ofsolids, gases and liquids in an attempt to discover ultrasoundcontrast-enhancing agents suitable for particular diagnostic purposes.Composite substances such as gelatin encapsulated microbubbles,gas-incorporated liposomes, sonicated partially denatured proteins andemulsions containing highly fluorinated organic compounds have also beenstudied in an attempt to develop an agent that has certain idealqualities, primarily, stability in the body and the ability to providesignificantly enhanced contrast in an ultrasound image.

Small bubbles of a gas, termed "microbubbles," are readily detected inan image produced using standard ultrasound imaging techniques. Wheninfused into the bloodstream or a particular site in the body,microbubbles enhance the contrast between the region containing themicrobubbles and the surrounding tissue.

A substantial amount of the research effort directed atcontrast-enhancing agents has focused on the use of extremely small gasbubbles. Investigators have long known that free gas bubbles provide ahighly effective contrast agent because a gas bubble has unique physicalcharacteristics that affect ultrasound energy as it is directed throughthe body. The advantages offered by free gas bubbles as opposed toliquid or solid agents that exhibit contrast enhancement is described indetail below in the context of the discussion of ultrasound diagnostictechniques.

Despite the known advantages, however, the rapid dissolution of free gasbubbles in solutions such as blood or many aqueous intravenoussolutions, severely limits their use as an ultrasound contrast-enhancingagent. The most important limitations are the size of the microbubbleand the length of time that a microbubble will exist before dissolvinginto the solution.

Examining the size requirements for microbubbles more closely, the gasbubbles must, of course, be sufficiently small that a suspension of thebubbles does not carry the risk of embolism to the organism in whichthey are infused. At the same time, extremely small free gas bubblescomposed of the gases generally used in ultrasound contrast imagingdissolve into solution so rapidly that their image-enhancing capabilityexists only immediately proximate to the infusion site. An additionalobstacle exists for ultrasound imaging of the cardiovascular system.Medical researchers have studied the time required for microbubblescomposed of ordinary air, pure nitrogen, pure oxygen, or carbon dioxide,to dissolve into solution. Microbubbles of these gases that aresufficiently small to be able to pass through the lungs and reach theleft heart, less than about 8 microns in diameter, have a life span ofless than approximately 0.25 seconds. Meltzer, R. S., Tickner, E. G.,Popp, R. L., "Why Do the Lungs Clear Ultrasonic Contrast?" Ultrasound inMedicine and Biology, Vol. 6, p.263, 267 (1980). Since it takes over 2seconds for blood to pass through the lungs, microbubbles of these gaseswould be fully dissolved during passage through the lungs and wouldnever reach the left heart. Ibid. Primarily because of this tradeoffbetween bubble size and life span, many researchers concluded that freegas microbubbles were not useful as a contract-enhancing agent forultrasound diagnosis of certain parts of the cardiovascular system.

However, the ultrasound contrast-enhancing media described hereincomprises microbubbles, composed of the gases whose selection is alsoprovided by this invention, are sufficiently small that they passthrough the pulmonary capillary diameter of approximately 8 microns andthereby allow contrast-enhanced ultrasound diagnosis of the leftchambers of the heart. The free gas microbubbles survive in thebloodstream long enough that they may be peripherally intravenouslyinfused, travel through the right heart, through the lungs, and into theleft cardiac chambers without dissolving into solution. Additionally,certain of these media have extremely long persistence in solution andwill enable contrast-enhancement of many other organs and structures.

This invention overcomes many of the inherent limitations thought toexist with the use of free gas microbubbles by providing, in part, amethod for selecting special gases based on particular physical criteriasuch that microbubbles composed of these gases do not suffer from thesame limitations as the microbubbles previously investigated. Therefore,it has been discovered that the ultrasound contrast-enhancing mediadescribed herein comprising a composition of microbubbles produced usinga gas or combination of gases selected by the physical and chemicalparameters disclosed herein can exist for a sufficient length of timeand be of sufficiently small size that their stability in thebloodstream allows enhanced ultrasound contrast imaging of particularstructures in the body previously thought inaccessible to free gasmicrobubbles.

Techniques For Measuring Ultrasound Contrast-Enhancement Phenomena

To more fully appreciate the subject matter of the present invention, itis useful to describe what is presently known about the technology ofultrasound imaging and to review the search for improved ultrasoundcontrast-enhancing agents in that light.

Materials that are useful as ultrasound contrast agents operate byhaving an effect on ultrasound waves as they pass through the body andare reflected to create the image from which a medical diagnosis ismade. In an attempt to develop an efficient image-contrast agent, oneskilled in the art recognizes that different types of substances affectultrasound waves in different ways and to varying degrees. Moreover,certain of the effects caused by contrast-enhancing agents are morereadily measured and observed than others. Thus, in selecting an idealcomposition for a contrast-enhancing agent, one would prefer thesubstance that has the most dramatic effect on the ultrasound wave as itpasses through the body. Also, the effect on the ultrasound wave shouldbe easily measured. There are three main contrast-enhancing effectswhich can be seen in an ultrasound image: backscatter, beam attenuation,and speed of sound differential. Each of these effects will be describedin turn.

A. BACKSCATTER

When an ultrasound wave that is passing through the body encounters astructure, such as an organ or other body tissue, the structure reflectsa portion of the ultrasound wave. Different structures within the bodyreflect ultrasound energy in different ways and in varying strengths.This reflected energy is detected and used to generate an image of thestructures through which the ultrasound wave has passed. The term"backscatter" refers to the phenomena in which ultrasound energy isscattered back towards the source by a substance with certain physicalproperties.

It has long been recognized that the contrast observed in an ultrasoundimage may be enhanced by the presence of substances known to cause alarge amount of backscatter. When such a substance is administered to adistinct part of the body, the contrast between the ultrasound image ofthis part of the body and the surrounding tissues not containing thesubstance is enhanced. It is well understood that, due to their physicalproperties, different substances cause backscatter in varying degrees.Accordingly, the search for contrast-enhancing agents has focused onsubstances that are stable and non-toxic and that exhibit the maximumamount of backscatter.

Making certain assumptions about the way a substance reflects ultrasoundenergy, mathematical formulae have been developed that describe thebackscatter phenomenon. Working with these formulae, a skilledresearcher can estimate the ability of gas, liquid, and solidcontrast-enhancing agents to cause backscatter and the degree to which aparticular substance causes measurable backscatter can be compared withother substances based on the physical characteristics known to causethe backscatter phenomenon. As a simple example, the ability ofsubstance A to cause backscatter will be greater than substance B, if,all other factors being equal, substance A is larger than substance B.Thus, when both substances are encountered by an ultrasound wave, thelarger substance scatters a greater amount of the ultrasound wave.

The capability of a substance to cause backscatter of ultrasound energyalso depends on other characteristics of the substance such as itsability to be compressed. Of particular importance is the dramaticincrease in backscatter caused by gas bubbles due to the bubbleresonance phenomenon which is described below. When examining differentsubstances, it is useful to compare one particular measure of theability of a substance to cause backscatter known as the "scatteringcross-section."

The scattering cross-section of a particular substance is proportionalto the radius of the scatterer, and also depends on the wavelength ofthe ultrasound energy and on other physical properties of the substance,J. Ophir and K. J. Parker, Contrast Agents in Diagnostic Ultrasound,Ultrasound in Medicine & Biology, Vol. 15, n. 4, p. 319, 323 (1989).

The scattering cross-section of a small scatterer, α, can be determinedby a known equation: ##EQU1## where κ=2π/λ, where λ is the wavelength;a=the radius of the scatterer; κ_(s) =adiabatic compressibility of thescatterer; κ=adiabatic compressibility of the medium in which thescatterer exists, ρ_(s) =density of the scatterers and ρ=the density ofthe medium in which the scatterer exists. P. M. Morse and K. U. Ingard,Theoretical Acoustics, p. 427, McGraw Hill, New York (1968).

In evaluating the utility of different substances as image contrastingagents, one can use this equation to determine which agents will havethe higher scattering cross-section and, accordingly, which agents willprovide the greatest contrast in an ultrasound image.

Referring to the above equation, the first bracketed quantity in theabove equation can be assumed to be constant for the purpose ofcomparing solid, liquid and gaseous scatterers. It can be assumed thatthe compressibility of a solid particle is much less than that of thesurrounding medium and that the density of the particle is much greater.Using this assumption, the scattering cross section of a solid particlecontrast-enhancing agent has been estimated as 1.75. Ophir and Parker,supra, at 325.

For a pure liquid scatterer, the adiabatic compressibility and densityof the scatterer κ_(s) and the surrounding medium κare likely to beapproximately equal which would, from the above equation, yield theresult that liquids would have a scattering cross-section of zero.However, liquids may exhibit some backscatter if large volumes of aliquid agent are present presumably because the term α in the firstbracketed quantity in the above equation may become sufficiently large.For example, if a liquid agent passes from a very small vessel to a verylarge one such that the liquid occupies substantially all of the vesselthe liquid may exhibit measurable backscatter. Nevertheless, in light ofthe above equation and the following, it is appreciated by those skilledin the art that pure liquids are relatively inefficient scattererscompared to free gas microbubbles.

It is known that changes in the acoustic properties of a substance arepronounced at the interface between two phases, i.e. liquid/gas, becausethe reflection characteristics of an ultrasound wave change markedly atthis interface. Additionally, the scatter cross-section of a gas issubstantially different than a liquid or solid, in part, because a gasbubble can be compressed to a much greater degree than a liquid orsolid. The physical characteristics of gas bubbles in solution are knownand standard values for compressibility and density figures for ordinaryair can be used in the above equation. Using these standard values, theresult for the second bracketed term alone in the above equation isapproximately 10¹⁴, Ophir and Parker supra, at 325, with the totalscattering cross section varying as the radius α of the bubble varies.Moreover, free gas bubbles in a liquid exhibit oscillatory motion suchthat, at certain frequencies, gas bubbles will resonate at a frequencynear that of the ultrasound waves commonly used in medical imaging. As aresult, the scattering cross-section of a gas bubble can be over athousand times larger than its physical size.

Therefore, it is recognized that gas micro-bubbles are superiorscatterers of ultrasound energy and would be an ideal contrast-enhancingagent if the obstacle of their rapid dissolution into solution could beovercome.

B. BEAM ATTENUATION

Another effect which can be observed from the presence of certain solidcontrast-enhancing agents, is the attenuation of the ultrasound wave.Image contrast has been observed in conventional imaging due tolocalized attenuation differences between certain tissue types. K. J.Parker and R. C. Wang, "Measurement of Ultrasonic Attenuation WithinRegions Selected from B-Scan Images," IEEE Trans. Biomed. Engr. BME30(8), p. 431-37 (1983); K. J. Parker, R. C. Wang, and R. M. Lerner,"Attenuation of Ultrasound Magnitude and Frequency Dependence for TissueCharacterization," Radiology, 153(3), p. 785-88 (1984). It has beenhypothesized that measurements of the attenuation of a region of tissuetaken before and after infusion of an agent may yield an enhanced image.However, techniques based on attenuation contrast as a means to measurethe contrast enhancement of a liquid agent are not well-developed and,even if fully developed, may suffer from limitations as to the internalorgans or structures with which this technique can be used. For example,it is unlikely that a loss of attenuation due to liquid contrast agentscould be observed in the image of the cardiovascular system because ofthe high volume of liquid contrast agent that would need to be presentin a given vessel before a substantial difference in attenuation couldbe measured.

Measurement of the attenuation contrast caused by microspheres ofAlbunex (Molecular Biosystems, San Diego, Calif.) in vitro has beenaccomplished and it has been suggested that in vivo attenuation contrastmeasurement could be achieved. H. Bleeker, K. Shung, J. Burnhart, "Onthe Application of Ultrasonic Contrast Agents for Blood Flowometry andAssessment of Cardiac Perfusion," J. Ultrasound Med. 9:461-471 (1990).Albunex is a suspension of 2-4 micron encapsulated air-filledmicrospheres that have been observed to have acceptable stability invivo and are sufficiently small in size that contrast enhancement canoccur in the left atrium or ventricle. Also, attenuation contrastresulting from iodipamide ethyl ester (IDE) particles accumulated in theliver has been observed. Under such circumstances, the contrastenhancement is believed to result from attenuation of the ultrasoundwave resulting from the presence of dense particles in a soft medium.The absorption of energy by the particles occurs by a mechanism referredto as "relative motion." The change in attenuation caused by relativemotion can be shown to increase linearly with particle concentration andas the square of the density difference between the particles and thesurrounding medium. K. J. Parker, et al., "A Particulate Contrast Agentwith Potential for Ultrasound Imaging of Liver," Ultrasound in Medicine& Biology, Vol. 13, No. 9, p. 555, 561 (1987). Therefore, wheresubstantial accumulation of solid particles occurs, attenuation contrastmay be a viable mechanism for observing image contrast enhancementalthough the effect is of much smaller magnitude than the backscatterphenomenon and would appear to be of little use in cardiovasculardiagnoses.

C. SPEED OF SOUND DIFFERENTIAL

An additional possible technique to enhance contrast in an ultrasoundimage has been proposed based on the fact that the speed of sound variesdepending on the media through which it travels. Therefore, if a largeenough volume of an agent, through which the speed of sound is differentthan the surrounding tissue, can be infused into a target area, thedifference in the speed of sound through the target area may bemeasurable. Presently, this technique is only experimental.

Therefore, considering the three techniques described above for contrastenhancement in an ultrasound image, the marked increase in backscattercaused by free gas microbubbles is the most dramatic effect andcontrast-enhancing agents that take advantage of this phenomenon wouldbe the most desirable if the obstacle of their limited stability insolution could be overcome.

The Materials Presently Used as Contrast-Enhancing Agents

In light of what is known about the various techniques described above,attempts to develop a contrast-enhancing agent whose presence generatessubstantial contrast in an ultrasound image, and whose survival in vivois sufficiently long to allow contrast-enhanced imaging of thecardiovascular system, has led to the investigation of a broad varietyof substances--gases, liquids, solids, and combinations of these--aspotential contrast-enhancing agents.

A. SOLID PARTICLES

Typically, the solid substances that have been studied as potentialcontrast-enhancing agents are extremely small particles that aremanufactured in uniform size. Large numbers of these particles can beinfused and circulate freely in the bloodstream or they may be injectedinto a particular structure or region in the body.

IDE particles are solid particles that can be produced in largequantities with a relatively narrow size distribution of approximately0.5-2.0 microns. Sterile saline injections of these particles may beinjected and will tend to accumulate in the liver. Once a substantialaccumulation occurs, contrast enhancement may be exhibited by eitherattenuation contrast or backscatter mechanisms. Although suspensionscomprising these solid particles dispersed in a liquid may exhibitacceptable stability, the backscatter or attenuation effects arerelatively minor compared to free gas bubbles and a substantialaccumulation of the particles must occur before appreciable contrast isobserved in an ultrasound image. Thus, use of these suspensions has beenlimited to certain cell types in which the particles have the tendencyto coagulate because unless the suspension becomes highly concentratedin particular tissue, the contrast enhancement will be minor.

SHU-454 (Schering, A. G., West Berlin, Germany) is an experimentalcontrast-enhancing agent in powder form that, when mixed with asaccharide diluent, forms a suspension of crystals of various rhomboidand polyhedral shapes ranging in size from 5 to 10 microns. Although theprecise mechanism by which these crystals enhance ultrasound contrast isnot completely understood, it is suspected that the crystals may trapmicrobubbles in their structure or that the crystals themselves maybackscatter ultrasound energy by an as-yet undetermined mechanism.

B. LIQUIDS AND EMULSIONS

In another attempt to achieve a satisfactory agent, emulsions areprepared by combining a chemical species compatible with body tissue anda species that provides high ultrasound contrast enhancement. EuropeanPatent Application 0231091 discloses emulsions of oil in watercontaining highly fluorinated organic compounds that have been studiedin connection with their possible use as a blood substitute and are alsocapable of providing enhanced contrast in an ultrasound image.

Emulsions containing perfluorooctyl bromide (PFOB) have also beenexamined. Perfluorooctyl bromide emulsions are liquid compounds known tohave the ability to transport oxygen. PFOB emulsions have exhibited alimited utility as ultrasound contrast agents because of a tendency toaccumulate in certain types of cells. Although the mechanism is notcompletely understood, PFOB emulsions may provide ultrasound contrastbecause of their high density and relatively large compressibilityconstant.

U.S. Pat. No. 4,900,540 describes the use of phospholipid-basedliposomes containing a gas or gas precursor as a contrast-enhancingagent. A liposome is a microscopic, spherical vesicle, containing abilayer of phospholipids and other amphipathic molecules and an inneraqueous cavity, all of which is compatible with the cells of the body.In most applications, liposomes are used as a means to encapsulatebiologically active materials. The above reference discloses the use ofa gas or gas precursors incorporated into the liposome core to provide alonger life span for the gas when infused into the body. Production ofstable liposomes is an expensive and time consuming process requiringspecialized raw materials and equipment.

C. MICROBUBBLES

As noted above, a critical parameter that must be satisfied by amicrobubble used as a contrast-enhancing agent is size. Free gasmicrobubbles larger than approximately 8 microns may still be smallenough to avoid impeding blood flow or occluding vascular beds. However,microbubbles larger than 8 microns are removed from the bloodstream whenblood flows through the lungs. As noted above, medical researchers havereported in the medical literature that microbubbles small enough topass through the lungs will dissolve so quickly that contrastenhancement of left heart images is not possible with a free gasmicrobubble. Meltzer, R. S., Tickner, E. G., Popp, R. L., "Why Do theLungs Clear Ultrasonic Contrast?" Ultrasound in Medicine and Biology,Vol. 6, p.263, 267 (1980).

However, cognizant of the advantages to be gained by use of microbubblesas contrast-enhancing agents by virtue of their large scatteringcross-section, considerable attention has been focused on developingmixtures containing microbubbles that are rendered stable in solution.Enhancing the stability of gas microbubbles may be accomplished by anumber of techniques.

Each of the following techniques essentially involves suspending acollection of microbubbles in a substrate in which a bubble of ordinarygas is more stable than in the bloodstream.

In one approach, microbubbles are created in viscous liquids that areinjected or infused into the body while the ultrasound diagnosis is inprogress. The theory behind the use of viscous fluids involves anattempt to reduce the rate at which the gas dissolves into the liquidand, in so doing, provide a more stable chemical environment for thebubbles so that their lifetime is extended.

Several variations on this general approach have been described. EPOApplication No. 0324938 describes a viscous solution of a biocompatiblematerial, such as a human protein, in which microbubbles are contained.By submitting a viscous protein solution to sonication, microbubbles areformed in the solution. Partial denaturation of the protein by chemicaltreatment or heat provides additional stability to microbubbles in thesolution by decreasing the surface tension between bubble and solution.

Therefore, the above approaches may be classified as an attempt toenhance the stability of microbubbles by use of a stabilizing medium inwhich the microbubbles are contained. However, none of these approacheshave addressed the primary physical and chemical properties of gaseswhich have seriously limited the use of free gas microbubbles inultrasound diagnosis, particularly with respect to the cardiovascularsystem. None of these approaches suggest that selection of the gases, byprecise criteria, would yield the ability to produce stable microbubblesat a size that would allow transpulmonary contrast-enhanced ultrasoundimaging.

The behavior of microbubbles in solution can be described mathematicallybased on certain parameters and characteristics of the gas of which thebubble is formed and the solution in which the bubble is present.Depending on the degree to which a solution is saturated with the gas ofwhich the microbubbles are formed, the survival time of the microbubblescan be calculated. P. S. Epstein, M. S. Plesset, "On the Stability ofGas Bubbles in Liquid-Gas Solutions," The Journal of Chemical Physics,Vol 18, n. 11, 1505 (1950). Based on calculations, it is apparent thatas the size of the bubble decreases, the surface tension between bubbleand surrounding solution increases. As the surface tension increases,the rate at which the bubble dissolves into the solution increasesrapidly and, therefore, the size of the bubble decreases more and morerapidly. Thus, the rate at which the bubble shrinks increases as thesize of the bubble decreases. The ultimate effect of this is that apopulation of small free gas microbubbles composed of ordinary airdissolves so rapidly that the contrast-enhancing effect is extremelyshort lived. Using known mathematical formula, one can calculate that amicrobubble of air that is 8 microns in diameter, which is small enoughto pass through the lungs, will dissolve in between 190 and 550milliseconds depending on the degree of saturation of the surroundingsolution. Based on these calculations, medical investigators studyingthe way in which the lungs remove ultrasound contrast agent havecalculated the dissolution times of oxygen and nitrogen gas microbubblesin human and canine blood and have concluded that free gas microbubblecontrast agents will not allow contrast-enhanced imaging of the leftventricle because of the extremely brief life of the microbubbles.

The physical properties of systems that feature gas bubbles or gasesdissolved in liquid solutions have been investigated in detail includingthe diffusion of air bubbles formed in the cavitating flow of a liquidand the scatter of light and sound in water by gas bubbles.

The stability of gas bubbles in liquid-gas solution has beeninvestigated both theoretically, Epstein P. S. and Plesset M. S., On theStability of Gas Bubbles in Liquid-gas Solutions, J. Chem. Phys.18:1505-1509 (1950) and experimentally, Yang W J, Dynamics of GasBubbles in Whole Blood and Plasma, J. Biomech 4:119-125 (1971); Yang WJ, Echigo R., Wotton D R, and Hwang J B, Experimental Studies of theDissolution of Gas Bubbles in Whole Blood and Plasma-I. StationaryBubbles. J. Biomech 3:275-281 (1971); Yang W J, Echigo R., Wotton D R,Hwang J B, Experimental Studies of the Dissolution of Gas Bubbles inWhole Blood and Plasma-II. Moving Bubbles or Liquids. J. Biomech4:283-288 (1971). The physical and chemical properties of the liquid andthe gas determine the kinetic and thermodynamic behavior of the system.The chemical properties of the system which influence the stability of abubble, and accordingly the life time, are the rate and extent ofreactions which either consume, transform, or generate gas molecules.

For example, a well known reaction that is observed between a gas and aliquid takes place when carbon dioxide gas is present in water. As thegas dissolves into the aqueous solution, carbonic acid is created byhydration of the carbon dioxide gas. Because carbon dioxide gas ishighly soluble in water, the gas diffuses rapidly into the solution andthe bubble size diminishes rapidly. The presence of the carbonic acid inthe solution alters the acid-base chemistry of the aqueous solution and,as the chemical properties of the solution are changed by dissolution ofthe gas, the stability of the carbon dioxide gas bubbles changes as thesolution becomes saturated. In this system, the rate of dissolution of agas bubble depends in part on the concentration of carbon dioxide gasthat is already dissolved in solution.

However, depending on the particular gas or liquid present in thesystem, the gas may be substantially insoluble in the liquid anddissolution of a gas bubble will be slower. In this situation, it hasbeen discovered that it is possible to calculate bubble stability in agas-liquid system by examining certain physical parameters of the gas.

BRIEF DESCRIPTION OF THE INVENTION

It has been discovered that it is possible to identify chemical systemswhere extremely small gas bubbles are not reactive in an aqueoussolution. Relying on the method disclosed herein one skilled in the artmay specially select particular gases based on their physical andchemical properties for use in ultrasound imaging. These gases can beused to produce the contrast-enhancing media that is also the subjectmatter of this invention. The microbubbles can be produced using certainexisting techniques that use ordinary air, and can be infused as in aconventional ultrasound diagnosis.

The method that is the subject matter of this invention requires thatcalculations be made, consistent with the equations provided herein,based on the intrinsic physical properties of a gas and a liquid.Particularly, the density of a gas, the solubility of a gas in solution,and the diffusivity of a gas in solution, which in turn is dependent onthe molar volume of the gas and the viscosity of the solution, are usedin the equations disclosed below. Thus, by the method disclosed herein,the physical properties of a given gas-liquid system can be evaluated,the rate and extent of bubble collapse can be estimated, and gases thatwould constitute effective contrast-enhancing agents can be selectedbased on these calculations. Using existing techniques, substantiallyimproved contrast-enhancing media may then be produced and used toimprove the quality and usefulness of ultrasound imaging.

DETAILED DESCRIPTION OF THE INVENTION

To understand the method of this invention, it is useful to derive themathematical relationships that describe the parameters of a gas-liquidsystem and the effect on bubble stability that occurs when a value forone or more of these parameters is altered. It is assumed that, at aninitial time, T_(o), a spherical gas bubble of gas X, with a radius ofR_(o), is placed in a solution in which the initial concentration of gasX dissolved in the solution is equal to zero. Over some period of time,the bubble of gas X will dissolve into the solvent at which time itsradius R will equal zero. Assume further that the solution is atconstant temperature and pressure and that the dissolved gasconcentration for a solution saturated with the particular gas isdesignated C_(s). Thus, at T_(o), the concentration of the gas in thesolution is zero, meaning that none of the gas has yet dissolved and allof the gas that is present is still contained within the bubble ofradius R_(o).

As time progresses, due to the difference in the concentration of thegas in the bubble and the gas in solution, the bubble will tend toshrink as gas in the bubble is dissolved into the liquid by the processof diffusion. The change in bubble radius from its original radius ofR_(o) to, after the passage of a particular amount of time t, a smallerradius R is expressed by Equation (1), ##EQU2## where R is the bubbleradius at time t, D is the coefficient of diffusivity of the particulargas in the liquid, and ρ is the density of the particular gas of whichthe bubble is composed.

It follows that the time T required for a bubble to dissolve completelymay be determined from Equation (1) by setting R/R_(o) =0, and solvingfor T: ##EQU3##

This result qualitatively indicates that bubble stability, and hencelife span, is enhanced by increasing the initial bubble size R_(o) or byselecting a gas of higher density ρ, lower solubility C_(s) in theliquid phase, or a lower coefficient of diffusivity D.

The diffusivity D of a gas in a liquid is dependent on the molar volumeof the gas (Vm), and the viscosity of the solution (η) as expressed by aknown Equation;

    D=13.26×10.sup.-5 ·η.sup.-1.14 ·V.sub.m.sup.-0.589                              (Equation 3)

By substituting the expression for D given in Equation (3) into Equation(2), it is revealed that bubble stability is enhanced by using gases oflarger molar volume Vm, which tend to have a higher molecular weight,and liquids of higher viscosity.

By way of example, a comparison of the stability of air microbubbles andmicrobubbles composed of gases specially selected by the methoddisclosed herein may be made. Taking the value of D for air in water at22° C. as 2×10⁻⁵ cm² sec⁻¹ and the ratio C_(s) /ρ=0.02 (Epstein andPlesset, Ibid.), one obtains the following data for the time t forcomplete solution of air bubbles in water (unsaturated with air):

                  TABLE I                                                         ______________________________________                                        INITIAL BUBBLE DIAMETER, microns                                                                     TIME, milliseconds                                     ______________________________________                                        12                     450                                                    10                     313                                                    8                      200                                                    6                      113                                                    5                      78                                                     4                      50                                                     3                      28                                                     2                      13                                                     1                       3                                                     ______________________________________                                    

If the blood transit time from the pulmonary capillaries to the leftventricle is two seconds or more (Hamilton, W. F. editor, Handbook ofPhysiology, Vol. 2, section 2, CIRCULATION. American Physiology Society,Washington, D.C., p. 709, (1963)), and recognizing that onlymicrobubbles of approximately 8 microns or less will be small enough topass through the lungs, it is clear that none of these bubbles have alife span in solution long enough to be useful contrast agents forultrasound contrast-enhanced imaging of the left ventricle.

The method of the present invention allows identification of potentiallyuseful gases by comparing the properties of any particular gas, denotedgas X in the following description, to air. Taking Equations (2) and (3)above, a coefficient Q may be formulated for a particular gas X thatwill describe the stability of microbubbles composed of gas. X in agiven liquid. The value of the Q coefficient determined by this methodfor a particular gas X also can be used to determine the utility of gasX as an ultrasound contrast-enhancing agent as compared to ordinary air.

From Equation (2) above, an equation that describes the time forcomplete dissolution of a bubble of gas X compared to the same sizebubble of ordinary air under identical conditions of solutiontemperature and solution viscosity may be written based on the physicalproperties of gas X and air: ##EQU4## or, if D is known for gas X,##EQU5## To formulate this equation so that the value Q may be obtainedto enable comparison of gas X with air, the above equation may berewritten: ##EQU6## Assuming for comparison, a solution of water at 22degrees C., the density, diffusivity, and solubility of air in thesolution are known quantities which may be substituted into the aboveequation yielding: ##EQU7## Substituting Equation (3) into the above forgases whose diffusivity Dx is not readily known, and assuming that theviscosity term η below for water at 22 degrees C. is approximately equalto 1.0 cP, ##EQU8## Thus, knowing the density, solubility and molarvolume of a gas, this method allows the calculation of the value of theQ coefficient.

If Q is less than one, microbubbles of gas X will be less stable in agiven solvent than microbubbles of air. If Q is greater than one,microbubbles formed of gas X are more stable than microbubbles of airand will survive in solution longer than air bubbles. All otherproperties being the same for a given microbubble size, the time forcomplete dissolution of a microbubble of gas X is equal to the time forcomplete dissolution of a microbubble of ordinary air multiplied by theQ coefficient. For example, if the Q coefficient for gas X is 10,000, amicrobubble of gas X will survive 10,000 times as long in solutioncompared to a microbubble of air. A Q value can be determined for anygas in any solution assuming the quantities identified herein are knownor can be estimated.

Different methods for determining or estimating values for theindividual parameters of density, diffusivity, and solubility may beneeded depending on the chemical structure of the gas. Values for theseparameters may or may not be available from known scientific literaturesources such as the Gas Encyclopaedia or the tabulations published bythe American Chemical Society. Values for the density of most gases arereadily available from sources such as the Handbook of Chemistry andPhysics, CRC Press, 72d Ed. (1991-92). Additionally, the solubility inwater and molar volume of some gases has been measured with accuracy. Inmany cases however, calculations for the numerical values for molarvolume and solubility may need to be calculated or estimated to providethe data used to determine the value of the Q coefficient for anindividual gas by the method described above. An example of thecalculation of Q values for a preferred selection of fluorocarbon gasesillustrates how the method of this invention can be applied toindividual gases.

EXAMPLE

Generally, fluorocarbon gases exhibit extremely low solubility in water,and have high molecular weights, high molar volumes, and high densities.To determine the Q value for several fluorocarbon gases, the solubility,molar volume and density of the individual gases are determined and thevalues are substituted into Equations (7) or (8) above.

Determination of Gas Solubility for Fluorocarbons

This method for estimating the gas solubility of fluorocarbons usesextrapolation of the experimental data of Kabalnov AS, Makarov KN, andScherbakova OV. "Solubility of Fluorocarbons in Water as a Key ParameterDetermining Fluorocarbon Emulsion Stability," J. Fluor. Chem. 50,271-284, (1990). The gas solubility of these fluorocarbons is determinedrelative to perfluoro-n-pentane which has a water solubility of 4.0×10⁻⁶moles per liter. For a homologous series of non-branched fluorocarbons,the gas solubility may be estimated by increasing or reducing this valueby a factor of about 8.0 for each increase or reduction in the number ofadditional --CF₂ -- groups present in the molecule.

Determination of Molar Volume

The molar volume (Vm) is estimated from the data of Bondi A., "Van derWaals Volumes and Radii," J. Phys. Chem. 68, 441-451 (1964). The molarvolume of a gas can be estimated by identifying the number and type ofatoms that make up the molecule of gas in question. By determining thenumber and type of atoms present in the molecule and how the individualatoms are bound to each other, known values may be applied for themolecular volume of the individual atoms. By considering thecontribution of each individual atom and its frequency of occurrence,one may calculate the total molar volume for a particular gas molecule.This calculation is best demonstrated with an example.

It is known that a carbon molecule in an alkane carbon-carbon bond has amolar volume of 3.3 cubic centimeters per mole, a carbon atom in analkene carbon-carbon bond has a molar volume of 10.0 cubic centimetersper mole, and when multiple fluorine atoms are bound to an alkanecarbon, a fluorine atom has a molar volume of 6.0 cubic centimeters permole.

Examining octafluoropropane, this molecule contains three carbon atomsin alkane carbon-carbon bonds (3 atoms at 3.3 cubic centimeters permole) and 6 fluorine atoms bound to alkane carbons (6 atoms at 6.0 cubiccentimeters per mole), hence, octafluoropropane has a molar density of58 cubic centimeters per mole.

Once density, molar volume, and solubility are determined, the Q valueis calculated using Equation 8 above.

The following Table lists the Q value for several fluorocarbon gasesbased on the calculations detailed above, the values for carbon dioxideare included for comparison.

                  TABLE II                                                        ______________________________________                                                            SOLU-     MOLAR                                                               BILITY    VOLUME                                                    DENSITY   micro-    cm.sup.3 /                                      GAS       kg/m.sup.3                                                                              moles/liter                                                                             mole    Q                                       ______________________________________                                        Carbon dioxide                                                                           1.977    33000     19.7     1                                      Sulfur     5.48      220      47      722                                     Hexafluoride                                                                  Hexa-     10(*)     2000      49      148                                     fluoropropylene                                                               Octa-     10.3       260      58      1299                                    fluoropropane                                                                 Hexa-      8.86     2100      43      116                                     fluoroethane                                                                  Octafluoro-2-                                                                           10(*)      220      65      1594                                    butene                                                                        Hexafluoro-2-                                                                            9(*)     2000      58      148                                     butyne                                                                        Hexafluorobuta-                                                                          9(*)     2000      56      145                                     1,3-diene                                                                     Octafluoro-                                                                              9.97      220      61      1531                                    cyclobutane                                                                   Deca-     11.21      32       73      13,154                                  fluorobutane                                                                  ______________________________________                                         *These density values are estimated from the known density of homologous      fluorocarbons.                                                           

Once the Q value has been determined the utility of an individual gas asan ultrasound contrast-enhancing agent can be analyzed by determiningthe life span of a collection of microbubbles composed of the gas inquestion at different sizes, as was done for air in Table 1 above.Taking the value of Q for decafluorobutane and examining the timenecessary for various sized bubbles to dissolve in water, one obtainsthe values in Table III below by multiplying each of the time values inTable I by the Q value for decafluorobutane:

                  TABLE III                                                       ______________________________________                                        INITIAL BUBBLE DIAMETER, microns                                                                     TIME, minutes                                          ______________________________________                                        12                     99                                                     10                     69                                                     8                      44                                                     6                      25                                                     5                      17                                                     4                      11                                                     3                      6.1                                                    2                      2.9                                                    1                      0.7                                                    ______________________________________                                    

Notice that the time scale in Table III is minutes rather thanmilliseconds as was the case for air. All bubbles of decafluorobutane,even as small as 1 micron, can be injected peripherally and will notdissolve into solution during the approximately 10 seconds needed toreach the left ventricle. Similar calculations can be performed for agas with any Q coefficient. Based on these calculations, a gas needs a Qvalue of at least 30 to comprise a useful agent for ultrasound contrastenhancement. Slightly larger bubbles will be able to pass through thelungs and yet survive long enough to permit both examination ofmyocardial perfusion and dynamic abdominal organ imaging. Moreover, aswith many of the gases identified by this method, decafluorobutanefeatures low toxicity at small dosages and would, therefore, offersubstantial advantages as a contrast-enhancing agent in conventionalultrasound diagnosis.

It will be appreciated by those skilled in the art that the lowsolubilities of these microbubbles in solution will require thatproduction of a suspension of these microbubbles be achieved by a methodwhich, suspends a quantity of gas into liquid rather than by a methodsuch as sonication which creates microbubbles from gases dissolved insolution. Manual creation of a microbubble suspension may beaccomplished by several methods. U.S. Pat. No. 4,832,941, the disclosureof which is incorporated herein by reference, refers to a method forproducing a suspension of microbubbles with a diameter less than sevenmicrons created by spraying a liquid through a quantity of gas using athree-way tap. Although techniques could vary in practice, the three-waytap is a preferred method to manually suspend a quantity of high Qcoefficient gas to produce the contrast-enhancing media describedherein.

The general techniques for use of a three-way tap device are well knownin connection with preparation of the common Freund's adjuvant forimmunizing research animals. Typically, a three-way tap is comprised ofa pair of syringes, both of which are connected to a chamber. Thechamber has an outlet from which the suspension may be collected orinfused directly.

Techniques for use of the three-way tap may differ from that describedin U.S. Pat. No. 4,832,941 because different gases are being used inthis procedure. For example, use of one of the high Q coefficient gasesdisclosed herein may be more efficient if the system is purged ofordinary air or flushed with another gas before the microbubblesuspension is produced.

In a preferred embodiment of the present invention, a 40-50% Sorbitol(D-glucitol) solution is mixed with approximately 1-10% by volume of ahigh Q-coefficient gas with approximately 5% gas being an optimal value.Sorbitol is a commercially available compound that when mixed in anaqueous solution substantially increases the viscosity of the solution.Higher viscosity solutions, as seen in equation 3 above, extend the lifeof a microbubble in solution. A 40-50% Sorbitol solution is preferred tomaintain as a bolus upon injection; that is as intact as possiblewithout exceeding a tolerable injection pressure. To produce thesuspension of microbubbles, a quantity of the chosen gas is collected ina syringe. In the same syringe, a volume of the Sorbitol solution may becontained. A quantity of Sorbitol solution is drawn into the othersyringe so that the sum of the two volumes yields the proper percentageof gas based on the volume percentage of microbubbles desired. Using thetwo syringes, each featuring a very small aperture, the liquid issprayed into the gas atmosphere approximately 25 times or as many timesas is necessary to create a suspension of microbubbles whose sizedistribution is acceptable for the purposes described herein. Thistechnique may be varied slightly, of course, in any manner that achievesthe resulting suspension of microbubbles of the desired size in adesired concentration. Microbubble size may be checked either visuallyor electronically using a Coulter Counter (Coulter Electronics) by aknown method, Butler, B. D., "Production of Microbubble for Use as EchoContrast Agents," J. Clin. Ultrasound, V.14 408 (1986).

Although the invention has been described in some respects withreference to specified preferred embodiments thereof, variations andmodifications will become apparent to those skilled in the art. It is,therefore, the intention that the following claims not be given arestrictive interpretation but should be viewed to encompass variationsand modifications that are derived from the inventive subject matterdisclosed.

I claim:
 1. Contrast media for ultrasound imaging comprising gaseousmicrobubbles selected from the group consisting of hexafluoropropylene,octafluoropropane, octafluoro-2-butene, hexafluoro-2-butyne,hexafluorobuta-1,3-diene, octafluorocyclobutane and decafluorobutane. 2.Contrast media of claim 1 comprising octafluoropropane.
 3. Contrastmedia of claim 1 comprising decafluorobutane.
 4. A biocompatibleultrasound contrast agent comprising perfluoropropane.
 5. The contrastagent of claim 4 wherein a portion of said perfluoropropane is presentas gaseous microbubbles suspended in a carrier.
 6. The contrast agent ofclaim 5 wherein said carrier is an aqueous liquid.
 7. The contrast agentof claim 4 wherein a portion of said microbubbles are less than 8microns in diameter.
 8. A biocompatible ultrasound contrast agentcomprising perfluorobutane.
 9. The contrast agent of claim 8 wherein aportion of said perfluorobutane is present as gaseous microbubblessuspended in a carrier.
 10. The contrast agent of claim 9 wherein saidcarrier is an aqueous liquid.
 11. The contrast agent of claim 8 whereina portion of said microbubbles are less than 8 microns in diameter. 12.A biocompatible ultrasound contrast agent containing gas-filledliposomes, the improvement comprising including microbubbles of at leastone gaseous fluorine-containing chemical in said liposomes.
 13. Thecontrast agent of claim 12 wherein the fluorine-containing chemical isselected from the group consisting of perfluoroethane, perfluoropropane,perfluorobutane, sulfur hexafluoride and mixtures thereof.
 14. Thecontrast agent of claim 12 wherein said liposomes are less than eightmicrons in diameter.
 15. A biocompatible ultrasound contrast agentcontaining a suspension of encapsulated air-filled microspheres, theimprovement comprising replacing all or a portion of the air with atleast one gaseous fluorine-containing chemical.
 16. The contrast agentof claim 15 wherein said microspheres are 2-4 microns in size.
 17. Thecontrast agent of claim 15 wherein the fluorine-containing chemical isselected from the group consisting of perfluoroethane, perfluoropropane,perfluorobutane, sulfur hexafluoride and mixtures thereof.
 18. Thecontrast agent of claim 15 wherein the microspheres are formed fromdenatured proteins.
 19. A biocompatible ultrasound contrast agentcontaining a suspension of crystals in a saccharide diluent, theimprovement comprising providing microbubbles of a perfluoroethane,perfluoropropane, perfluorobutane and sulfur hexafluoride.
 20. Thecontrast agent of claim 19 wherein said crystals range in size from 5 to10 microns.
 21. A biocompatible ultrasound contrast agent containing anemulsion of highly fluorinated organic compounds, the improvementcomprising providing microbubbles of at least one gaseous fluorinecontaining chemical in said emulsion.
 22. A biocompatible ultrasoundcontrast agent containing an air-filled microbubble suspension, theimprovement comprising providing perfluoroethane, perfluorobutane,perfluoropropane or sulfur hexafluoride gas within the microbubbles ofsaid suspension.